Multiprotein machines control many complex biological processes, such as DNA replication, RNA processing, and protein degradation. The 26S proteasome is responsible for the majority of protein degradation in eukaryotes and is essential for numerous cellular activities. Aberrant proteasomal activity impacts many human diseases including cancer, neurodegenerative disorders, and diabetes. Like many multisubunit catalytic complexes, the proteasome forms ring-shaped structures through which a substrate is passed, allowing multiple enzymatic activities to act sequentially on the substrate. The proteasome consists of a cylindrical 20S proteasome core particle (CP) with a 19S regulatory particle (RP) on one or both ends. Each RP can be divided into lid and base subcomplexes. The CP has two outer heteroheptameric 1 rings that sandwich a pair of 2 rings, which house the central proteolytic chamber. The RP base includes six different ATPases that form a hexameric ring; the RP binds and unfolds substrates and drives them into the CP interior. How these large complexes are assembled in vivo is poorly understood. This is true for the CP and even more so for the ~19-subunit RP. Proteasome assembly appears to be an ordered process conserved across species. As proteasomes are highly abundant, assembly must occur with high fidelity to avoid excessive accumulation of nonproductive and potentially toxic off-pathway intermediates. Both CP and RP assembly depend on dedicated assembly chaperones. The long-range goal of this grant is to delineate the pathways of eukaryotic 26S proteasome biogenesis. The proteasome has emerged as an important target for anti-cancer treatment and other therapies. Interfering with its assembly will provide a new approach for pharmaceutical intervention. The proposed experiments use a combination of genetic, biochemical, cell biological and biophysical methods and are focused primarily on the model eukaryote Saccharomyces cerevisiae, which has a 26S proteasome very similar to the human complex. A major goal of this first renewal will be on deciphering the assembly-promoting mechanisms of a CP assembly factor (Aim 1) and four RP assembly chaperones (RACs) (Aim 2). Both sets of factors were discovered during the past cycle of this grant. Aim 3 is directed toward determining what intermediates of the RP lid form in vivo and whether these serve as assembly intermediates. Lid-base joining will be a key step to be analyzed. Finally, Aim 4 considers the question of where in the cell the proteasome assembles and how proteasomes relocalize within the cell in response to changes in growth conditions. PUBLIC HEALTH RELEVANCE: Many human diseases result from an abnormally high or low level of some crucial protein that controls cell or tissue function. Some cancers, for instance, are caused by insufficient amounts of proteins called tumor suppressors. The proteasome is a huge enzyme complex that controls the levels of important proteins by degrading them; adjustment of proteasome assembly or activity can therefore have a profound impact on the course of many human disorders.